Ultra performance liquid chromatography (UPLC) is a new category of liquid chromatography that researchers are using to increase resolution, speed, and sensitivity in a variety of applications. These benefits result from packing columns with 1.7 μm particles and using instruments that are optimized for such columns.

Peptide mapping is a complex process that uses samples requiring shallow gradients with long run times. This article describes chemical and operating considerations when adapting UPLC to peptide mapping. At optimal separation conditions, UPLC peptide maps show more resolution than other methods and increased sensitivity. UPLC can generate particularly good peak shapes for glycopeptides, and find good resolution for deamidated peptides. The UPLC columns show significant increases in electrospray ionization mass spectrometry (ESI-MS) sensitivity, with only small loss of retention and peak shape when formic acid is used in place of trifluoroacetic acid (TFA). We assert that the compelling benefits of UPLC for peptide mapping suggest that it will become the technique of choice in the near future.

MAKING PEPTIDE MAPS

Peptide mapping is a workhorse technique in biopharmaceutical characterization.1 It is used to identify proteins based on the elution pattern of the peptide fragments, determine post-translational modifications, confirm genetic stability, and analyze protein sequence when interfaced with mass spectrometry.

To make a peptide map, it is necessary to separate every peptide into a single peak. Therefore, peptide mapping represents a significant chromatographic challenge, because of the complexity of peptide digests. In addition to the large number of peptides that are generated from the enzymatic digest of a protein, there can be a large number of alternative peptide structures, such as post-translational modifications.

There has been a long trend of reducing particle size in liquid chromatography. Modern reversed phase liquid chromatography (LC) began in the mid-1970s with the advent of irregular 10 μm particles, and within the last five years 2.5 μm particles have become available. The smaller particles have been used in short columns, which leads to fast analysis times but relatively modest gains in resolving power.

Column length has decreased with particle size because the system pressure required is inversely proportional to the cube of particle diameter. For example, reducing the particle size by a factor of two requires an increase in the operating pressure by a factor of eight. It is, therefore, necessary to use shorter columns at lower flow rates to remain within the capabilities of the instrument. Clearly, in order to take advantage of smaller particle sizes, both in terms of improved speed and improved resolution, we require instrumentation capable of high-pressure operation. In addition, system band broadening must be reduced to observe the narrow peaks generated with small particle packings.

In 2004, Schwartz and Murphy introduced the first LC system capable of operation up to 15,000 psi (1,000 bar).2 The combination of a system capable of high-pressure operation and columns packed with sub-2 μm particles has been termed ultra performance liquid chromatography (UPLC) to differentiate it from high performance LC (HPLC).3

Table 1. LC Conditions

The benefits of UPLC vs. HPLC were originally demonstrated for small molecules (<500 Da) with reversed phase columns. Improvements in resolving power (1.7x), sensitivity (3x) and separation speed (9x) were demonstrated for many different applications.3,4 All these benefits derive from the 1.7 μm particles used in UPLC columns. In addition to the high-pressure capabilities, the UPLC instrument also reduces system volume and detector-cell volume to preserve the high-efficiency separations. UPLC has gained rapid acceptance for small-molecule analytical separations, and we believe that it will displace HPLC for many applications. The capabilities of UPLC should make higher resolution peptide mapping possible.